1. Field of the Invention
This invention pertains to a sensor and method for detecting or quantifying analytes. More particularly the present invention is directed to the detection of analytes by interaction thereof with an immobilized macromolecular entity and the analysis of certain de novo electrical effects that are produced as a result of such interactions. The invention is an extension of the sensor and method described in PCT application PCT/IL99/00309 of common assignee herewith.
2. Description of the Related Art
Chemical and biological sensors are devices that can detect or quantify analytes by virtue of interactions between targeted analytes and macromolecular binding agents such as enzymes, receptors, DNA strands, heavy metal chelators, or antibodies. Such sensors have practical applications in many areas of human endeavor. For example, biological and chemical sensors have potential utility in fields as diverse as blood glucose monitoring for diabetics, detection of pathogens commonly associated with spoiled or contaminated food, genetic screening, and environmental testing.
Chemical and biological sensors are commonly categorized according to two features, namely, the type of material utilized as binding agent and the means for detecting an interaction between binding agent and targeted analyte or analytes. Major classes of biosensors include enzyme (or catalytic) biosensors, immunosensors and DNA biosensors. Chemical sensors make use of synthetic macromolecules for detection of target analytes. Some common methods of detection are based on electron transfer, generation of chromophores, or fluorophores, changes in optical or acoustical properties, or alterations in electric properties when an electrical signal is applied to the sensing system.
Enzyme (or catalytic) biosensors utilize one or more enzyme types as the macromolecular binding agents and take advantage of the complementary shape of the selected enzyme and the targeted analyte. Enzymes are proteins that perform most of the catalytic work in biological systems and are known for highly specific catalysis. The shape and reactivity of a given enzyme limit its catalytic activity to a very small number of possible substrates. Enzymes are also known for speed, working at rates as high as 10,000 conversions per second per enzyme molecule. Enzyme biosensors rely on the specific chemical changes related to the enzyme/analyte interaction as the means for determining the presence of the targeted analyte. For example, upon interaction with an analyte, an enzyme may generate electrons, a colored chromophore or a change in pH (due to release of protons) as the result of the relevant catalytic enzymatic reaction. Alternatively, upon interaction with an analyte, an enzyme may cause a change in a fluorescent or chemiluminescent signal that can be recorded by an appropriate detection system.
Immunosensors utilize antibodies as binding agents. Antibodies are protein molecules that bind with specific foreign entities, called antigens, that can be associated with disease states. Antibodies attach to antigens and either remove the antigens from a host and/or trigger an immune response. Antibodies are quite specific in their interactions and, unlike enzymes, they are capable of recognizing and selectively binding to very large bodies such as single cells. Thus, antibody-based biosensors allow for the identification of certain pathogens such as dangerous bacterial strains. As antibodies generally do not perform catalytic reactions, there is a need for special methods to record the moment of interaction between target analyte and recognition agent antibody. Changes in mass (surface plasmon resonance, acoustic sensing) are often recorded; other systems rely on fluorescent probes that give signals responsive to interaction between antibody and antigen. Alternatively, an enzyme bound to an antibody can be used to deliver the signal through the generation of color or electrons; the ELISA (Enzyme-Linked ImmunoSorbent Assay) is based on such a methodology.
DNA biosensors utilize the complimentary nature of the nucleic acid double-stands and are designed for the detection of DNA or RNA sequences usually associated with certain bacteria, viruses or given medical conditions. A sensor generally uses single-stands from a DNA double helix as the binding agent. The nucleic acid material in a given test sample is then denatured and exposed to the binding agent. If the strands in the test sample are complementary to the strands used as binding agent, the two interact. The interaction can be monitored by various means such as a change in mass at the sensor surface or the presence of a fluorescent or radioactive signal. Alternative arrangements have binding of the sample of interest to the sensor and subsequent treatment with labeled nucleic acid probes to allow for identification of the sequence(s) of interest.
Chemical sensors make use of non-biological macromolecules as binding agents. The binding agents show specificity to targeted analytes by virtue of the appropriate chemical functionalities in the macromolecules themselves. Typical applications include gas monitoring or heavy metal detection; the binding of analyte may change the conductivity of the sensor surface or lead to changes in charge that can be recorded by an appropriate field-effect transistor (FET). Several synthetic macromolecules have been used successfully for the selective chelation of heavy metals such as lead.
The present invention has applicability to all of the above noted binding agent classes.
Known methods of detecting interaction of analyte and binding agent can be grouped into several general categories: chemical, optical, acoustical, and electrical. In the last, a voltage or current is applied to the sensor surface or an associated medium. As binding events occur on the sensor surface, there are changes in electrical properties of the system. The leaving signal is altered as function of analyte presence.
The most relevant prior art to the present invention involves sensors that are based on electrical means for analyte detection. There are several classes of sensors that make use of applied electrical signals for determination of analyte presence. xe2x80x9cAmperometricxe2x80x9d sensors make use of oxidation-reduction chemistries in which electrons or electrochemically active species are generated or transferred due to analyte presence. An enzyme that interacts with an analyte may produce electrons that are delivered to an appropriate electrode; alternatively, an amperometric sensor may employ two or more enzyme species, one interacting with analyte, while the other generates electrons as a function of the action of the first enzyme (a xe2x80x9ccoupledxe2x80x9d enzyme system). Glucose oxidase has been used frequently in amperometric biosensors for glucose quantification for diabetics. Other amperometric sensors make use of electrochemically active species whose presence alters the system applied voltage as recorded at a given sensor electrode. Not all sensing systems can be adapted for electron generation or transfer, and thus many sensing needs cannot be met by amperometric methods alone. The general amperometric method makes use of an applied voltage and effects of electrochemically active species on said voltage. An example of an amperometric sensor is described in U.S. Pat. No. 5,593,852, in which Heller and Pishko disclose a glucose sensor that relies on electron transfer effected by a redox enzyme and electrochemically-active enzyme cofactor species. The present invention does not require application of an eternal voltage, oxidation/reduction chemistry, or electron generation/transfer.
An additional class of electrical sensing systems includes those sensors that make use primarily of changes in an electrical response of the sensor as a function of analyte presence. Some systems pass an electric current through a given medium; if analyte is present, there is a corresponding change in exit electrical signal, and this change implies that analyte is present. In some cases, the binding agent-analyte complex causes an altered signal, while in other systems, the bound analyte itself is the source of changed electrical response. Such sensors are distinguished from amperometric devices in that they do not necessarily require the transfer of electrons to an active electrode. Sensors based on the application of an electrical signal are not universal, in that they depend on alteration of voltage or current as a function of analyte presence; not all sensing systems can meet such a requirement. An example of this class of sensors is U.S. Pat. No. 5,698,089, in which Lewis and Freund disclose a chemical sensor in which analyte detection is determined by change of an applied electrical signal. Binding of analyte to chemical moieties arranged in an array alters the conductivity of the array points; unique analytes can be determined by the overall changes in conductivity of all of the array points. The present invention does not rely on arrays or changes of applied electrical signal as a function of analyte presence. The present sensor does not require any applied electrical or electromagnetic signal.
Several other publications that do not fall into the preceding categories are worthy of mention in the prior art. Radmacher, et al. (Science 265:1577-1579 (1994)) noted the existence of augmented spatial fluctuations in enzymes interacting with substrates, but did not apply this phenomenon to analyte detection. Holzrichter, et al., U.S. Pat. No. 5,620,854, did make use of macromolecule motion to detect analyte; their system relies specifically on atomic force or scanning tunneling microscopes for detection of said motion. An additional patent is that of Stanbro, et al., U.S. Pat. No. 5,114,674, which discloses a sensor that is based on the interference of applied electrical fields. Interaction of target analyte with a binding agent alters the interference of the applied electrical field. The present invention does not make use of applied electrical fields, currents, or voltages.
Other prior-art voltage-based sensors require the use of semiconducting field-effect transistors (FET""s) and rely on the chemical generation or physical trapping of charged species near the sensor surface. The method has found widespread use in the detection of positively-charged heavy metals as well as analytes that are involved in proton (H+) generating enzyme reactions. Sato, et al. (xe2x80x9cEndoscopic Urease Sensor System for Detecting Helicobacter pylori on Gastric Mucosaxe2x80x9d, Gastrointestinal Endoscopy 49: 32-38 (1999)) describe a pH-sensitive FET for the detection of the enzyme urease associated with the pathogenic bacteria H pylori. The present invention does not rely on the use of a FET in the sensor element contacted to analyte-containing samples.
While hundreds of sensors have been described in patents and in the scientific literature, actual commercial use of such sensors remains limited. In particular, virtually all sensor designs set forth in the prior art contain one or more inherent weaknesses. Some lack the sensitivity and/or speed of detection necessary to accomplish certain tasks. Other sensors lack long-term stability. Still others cannot be sufficiently miniaturized to be commercially viable or are prohibitively expensive to produce. Some sensors must be pre-treated with salts and/or enzyme cofactors, a practice that is inefficient and bothersome. To date, virtually all sensors are limited by the known methods of determining that contact has o between an immobilized binding agent and targeted analytes. Use of fluorescent or other external detection probes adds to sensor production requirements and reduces lifetimes of such sensor systems. Additionally, the inventor believes that there is no sensor method disclosed in the prior art that is general applicable to the vast majority of macromolecular binding agents, including enzymes, antibodies, antigens, nucleic acids, receptors, and synthetic binding agents.
It is therefore a primary object of the present invention to provide an improved analyte detection system, in which a detection unit is electrically connected to a sensor strip so as to allow for detection of internally-generated electrical signals in the sensor strip that are responsive to analyte presence;
It is a further object of the invention to describe an electrical circuit that includes sensor strip and detection unit to allow for rapid, sensitive, and inexpensive analyte detection; and,
It is an additional object of the invention to improve the consistency and ease of use in detection of an analyte in a sensor system by inclusion of resistance-modifying elements in the detector unit-sensor strip electrical circuit.
These and other objects of the present invention are attained by a sensor that includes a base member having a conductive electrical property; a macromolecular entity bound to the base member, wherein the macromolecular entity and the base member define a sensor strip, the macromolecular entity being interactive at a level of specificity with a predetermined analyte, and an electrical signal is internally-generated in the sensor strip responsive to presence of the analyte; and, a resistance-modifying element disposed in a circuit between the base member and a detection unit for detection of the electrical signal.
According to still another aspect of the invention, the resistance-modifying element is a self-assembled monolayer.
According to yet another aspect of the invention, the resistance-modifying element is a chemical entity.
According to another aspect of the invention, the internally-generated electrical signal is further processed for determination of analyte presence or concentration.
According to yet another aspect of the invention, a serial dilution unit is included for the determination of analyte concentration.
The invention provides a method for detecting analyte, including the following steps: providing an electrically conductive base member; immobilizing at least one macromolecule in proximity to the base member, wherein the macromolecule is capable of interacting at a level of specificity with a predetermined analyte, wherein the base member and the macromolecule define a sensor strip; exposing predetermined analyte to the macromolecule; and, detecting an electrical signal internally-generated in the sensor strip, the electrical signal being responsive to a presence of the predetermined analyte, wherein the step of detecting is performed with an electrical circuit that includes the base member and a resistance modifying element.
According to an aspect of the invention, contact between equipotential electrodes of a detection unit and the sensor strip is facilitated by presence of an adhesive agent between the sensor strip and detection unit electrodes.
According to an additional aspect of the invention, an additional step includes processing the electrical signals for determination of analyte presence.
According to still another aspect of the invention, the method includes the steps of serially diluting a sample and exposing the serial dilutions to a plurality of sensor strips for the determination of analyte concentration range.